1. Field of the Invention
The present invention is in the fields of protein biochemistry and the pharmaceutical and medical sciences. In particular, the invention provides methods for the production of conjugates between water-soluble polymers (e.g., poly(ethylene glycol) and derivatives thereof) and bioactive components, which conjugates have reduced antigenicity and immunogenicity compared to standard polymer-bioactive component conjugates. The invention also provides conjugates produced by such methods, compositions comprising such conjugates, kits comprising such conjugates and compositions and methods of use of the conjugates and compositions in preventing, diagnosing and treating a variety of medical and veterinary conditions.
2. Related Art
Two key factors have hindered the development of recombinant proteins as therapeutic agents—their generally short half-lives in the circulation and their potential antigenicity and immunogenicity. As used herein and generally in the art, the term “antigenicity” refers to the ability of a molecule to bind to preexisting antibodies, while the term “immunogenicity” refers to the ability to evoke an immune response in vivo, whether that response involves the formation of antibodies (a “humoral response”) or the stimulation of cellular immune responses. For the administration of recombinant therapeutic proteins, intravenous (i.v.) administration is often desirable in order to achieve the highest circulating activities and to minimize problems of bioavailability and degradation. However, the half-lives of small proteins following i.v. administration are usually extremely short (see examples in Mordenti, J., et al., (1991) Pharm Res 8:1351-1359; Kuwabara, T., et al., (1995) Pharm Res 12:1466-1469). Healthy kidneys generally retain in the bloodstream proteins with hydrodynamic radii exceeding that of serum albumin, which has a Stokes radius of c. 36 Å and a molecular weight of c. 66,000 Daltons (66 kDa). However, smaller proteins, such as granulocyte colony-stimulating factor (“G-CSF”) and ribonuclease, are cleared rapidly from the bloodstream by glomerular filtration (Brenner, B. M., et al. (1978) Am J Physiol 234:F455-F460; Venkatachalam, M. A., et al. (1978) Circ Res 43:337-347; Wilson, G., (1979) J Gen Physiol 74:495-509). As a result, maintenance of therapeutically useful concentrations of small recombinant proteins in the circulation is problematic following i.v. administration. Therefore, higher concentrations of such proteins and more frequent injections must be administered. The high dose rate increases the cost of therapy, decreases the likelihood of patient compliance and increases the risk of adverse events, e.g., immune reactions. Both cellular and humoral immune responses can reduce the circulating concentrations of injected recombinant proteins to an extent that may preclude the administration of an effective dose or may lead to treatment-limiting events such as anaphylaxis (Pui, C.-H., et al. (2001) J Clin Oncol 19:697-704).
Alternative routes of administration, such as subcutaneous (s.c.) or intramuscular (i.m.) injections, can overcome some of these problems, by providing more gradual release of recombinant proteins into the circulation. However, the bioavailability can be quite low, making it difficult to achieve effective circulating concentrations of such drugs. A further problem that may be related to the poor bioavailability of drugs administered s.c. or i.m. is the increased probability of degradation of the therapeutic protein at the site of injection.
Modification of recombinant proteins by the covalent attachment of derivatives of poly(ethylene glycol) (“PEG”) has been investigated extensively as a means of addressing the shortcomings discussed above (reviewed in Sherman, M. R., et al. (1997) in: Poly(ethylene glycol): Chemistry and Biological Applications, Harris, J. M., et al., eds., American Chemical Society, Washington, D.C., pp. 155-169; Roberts, M. J., et al. (2002) Adv Drug Deliv Res 54:459-476). The attachment of PEG derivatives to proteins has been shown to stabilize the proteins, improve their bioavailability and/or reduce their immunogenicity in vivo. (The covalent attachment of PEG derivatives to a protein or other substrate is referred to herein, and is known in the art, as “PEGylation.”) In addition, PEGylation can increase the hydrodynamic radius of proteins significantly. When a small protein, such as a cytokine or polypeptide hormone, is coupled to a single long strand of PEG (e.g., having a molecular weight of at least about 18 kDa), the resultant conjugate has a larger hydrodynamic radius than that of serum albumin and its clearance via the renal glomeruli is dramatically retarded. The combined effects of PEGylation—reduced proteolysis, reduced immune recognition and reduced rates of renal clearance—confer substantial advantages on PEGylated proteins as therapeutic agents.
Since the 1970s, attempts have been made to use the covalent attachment of polymers to improve the safety and efficacy of various proteins for pharmaceutical use (see, e.g., U.S. Pat. No. 4,179,337). Some examples include the coupling of PEG or poly(ethylene oxide) (PEO) to adenosine deaminase (EC 3.5.4.4) for use in the treatment of severe combined immunodeficiency disease (Davis, S., et al. (1981) Clin Exp Immunol 46:649-652; Hershfield, M. S., et al. (1987) N Engl J Med 316:589-596). Other examples include the coupling of PEG to superoxide dismutase (EC 1.15.1.1) for the treatment of inflammatory conditions (Saifer, M., et al., U.S. Pat. Nos. 5,006,333 and 5,080,891) and to urate oxidase (EC 1.7.3.3) for the elimination of excess uric acid from the blood and urine (Inada, Y., Japanese Patent Application 55-099189; Kelly, S. J., et al. (2001) J Am Soc Nephrol 12:1001-1009; Williams, L. D., et al., PCT publication WO 00/07629 A3, corresponding to U.S. Pat. No. 6,576,235; Sherman, M. R., et al., PCT publication WO 01/59078 A2).
PEOs and PEGs are polymers composed of covalently linked ethylene oxide units. These polymers have the following general structure:R1—(OCH2CH2)n—R2 where R2 may be a hydroxyl group (or a reactive derivative thereof) and R1 may be hydrogen, as in “PEG diol”, a methyl group, as in monomethoxyPEG (“mPEG”), or another lower alkyl group, e.g., as in iso-propoxyPEG or t-butoxyPEG. The parameter n in the general structure of PEG indicates the number of ethylene oxide units in the polymer and is referred to herein and in the art as the “degree of polymerization.” PEGs and PEOs can be linear, branched (Fuke, I., et al. (1994) J Control Release 30:27-34) or star-shaped (Merrill, E. W. (1993) J Biomater Sci Polym Ed 5:1-11). PEGs and PEOs are amphipathic, i.e. they are soluble in water and in certain organic solvents and they can adhere to lipid-containing materials, including enveloped viruses and the membranes of animal and bacterial cells. Certain random or block or alternating copolymers of ethylene oxide (OCH2CH2) and propylene oxide, which has the following structure:
have properties that are sufficiently similar to those of PEG that these copolymers are thought to be suitable replacements for PEG in certain applications (see, e.g., U.S. Pat. Nos. 4,609,546 and 5,283,317). The term “polyalkylene oxides” and the abbreviation “PAOs” are used herein to refer to such copolymers, as well as to PEG or PEO and poly(oxyethylene-oxymethylene) copolymers (U.S. Pat. No. 5,476,653). As used herein, the term “polyalkylene glycols” and the abbreviation “PAGs” are used to refer generically to polymers suitable for use in the conjugates of the invention, particularly PEGs, more particularly PEGs containing a single reactive group (“mono functionally activated PEGs”).
Commonly, several (e.g., 5 to 10) strands of one or more PAGs, e.g., one or more mPEGs with a molecular weight of about 5 kDa to about 10 kDa, are coupled to the target protein via primary amino groups (the epsilon amino groups of lysine residues and the alpha amino group of the N-terminal amino acid). More recently, conjugates have been synthesized containing a single strand of mPEG of higher molecular weight, e.g., 12 kDa, 20 kDa or 30 kDa. Direct correlations have been demonstrated between the plasma half-lives of the conjugates and an increasing molecular weight and/or increasing number of strands of PEG coupled (Clark, R., et al. (1996) J Biol Chem 271:21969-21977). On the other hand, as the number of strands of PEG is increased, so is the probability that an amino group in an essential region of the bioactive component (particularly if the bioactive component is a protein) will be modified, impairing its biological function (e.g., catalysis by an enzyme or receptor binding by a cytokine). For larger proteins that contain many amino groups, and for enzymes with substrates of low molecular weight, this tradeoff between increased duration of action and decreased specific activity may be acceptable, since it produces a net increase in the biological activity of the PEG-containing conjugates in vivo. For smaller proteins, such as polypeptide hormones and cytokines, however, a relatively high degree of substitution is likely to decrease the functional activity to the point of negating the advantage of an extended half-life in the bloodstream (Clark, R., et al., supra).
Certain of the present inventors have pioneered a variety of PEGylation strategies and have applied them to several proteins to achieve the desirable combination of favorable pharmacokinetics and increased potency in vivo. These proteins include granulocyte-macrophage colony-stimulating factor (“GM-CSF”) (Saifer, M. G. P., et al. (1997) Polym Preprints 38:576-577; Sherman, M. R., et al. (1997) supra) and recombinant mammalian uricase (see PCT publications WO 00/07629 and WO 01/59078; Kelly, S. J., et al., supra; U.S. Pat. No. 6,576,235). Using GM-CSF as a model cytokine, certain of the present inventors demonstrated that the attachment of one or two strands of MPEG of high molecular weight (about 36 kDa) was sufficient to enhance dramatically the potency of recombinant murine GM-CSF in vivo (Saifer, M. G. P., et al. (1997) supra; Sherman, M. R., et al. (1997) supra). Studies have also been conducted in which recombinant mammalian urate oxidase (uricase) was modified and explored as a potential treatment for intractable gout (see PCT publications WO 00/07629, corresponding to U.S. Pat. No. 6,576,235, and WO 01/59078, the disclosures of which are entirely incorporated herein by reference). When the PEG-uricase was used to treat uricase-deficient mice (uox −/−) that displayed profound uric acid-induced nephropathy, it was found to be well tolerated, effective and substantially non-immunogenic. Treated mice exhibited improved renal function for the duration of treatment (10 weeks) and had substantially less uric acid-related kidney damage than untreated uox −/− mice, as demonstrated by microscopic magnetic resonance imaging (Kelly, S. J., et al. (2001) supra).
The covalent attachment of strands of a PAG to a polypeptide molecule is disclosed in U.S. Pat. No. 4,179,337 to Davis, F. F., et al., as well as in Abuchowski, A., et al. (1981) in: Enzymes as Drugs, Holcenberg, J. S., et al., eds., John Wiley and Sons, New York, pp. 367-383. These references disclose that enzymes and other proteins modified with mPEGs have reduced immunogenicity and antigenicity and have longer lifetimes in the bloodstream, compared to the corresponding unmodified proteins. The resultant beneficial properties of the chemically modified conjugates are very useful in a variety of therapeutic applications.
To effect the covalent attachment of PEG or polyalkylene oxides to a protein, at least one of the hydroxyl end groups of the polymer must first be converted into a reactive functional group. This process is frequently referred to as “activation” and the product is called “activated PEG” or activated polyalkylene oxide. MonomethoxyPEG that is capped on one end with an unreactive, chemically stable methyl ether (the “methoxyl group”) and on the other end with a functional group reactive towards amino groups on a protein molecule is most commonly used for such approaches. So-called “branched” mPEGs, which contain two or more methoxyl groups distal from a single activated functional group, are used less commonly. An example is di-mPEG-lysine, in which the carboxyl group of lysine is most often activated by esterification with N-hydroxysuccinimide (Harris, J. M., et al., U.S. Pat. No. 5,932,462).
The activated polymers are reacted with a therapeutic agent having nucleophilic functional groups that serve as attachment sites. One nucleophilic functional group commonly used as an attachment site is the epsilon amino group of lysine residues. Free carboxylic acid groups, suitably activated carbonyl groups, oxidized carbohydrate moieties and thiol groups have also been used as attachment sites.
The hydroxyl group of MPEG has been activated with cyanuric chloride and the resulting compound then coupled with proteins (Abuchowski, A., et al. (1977) J Biol Chem 252:3582-3586; Abuchowski, A., et al. (1981) supra). The use of this method has disadvantages however, such as the toxicity of cyanuric chloride and its non-specific reactivity for proteins having functional groups other than amines, such as solvent-accessible cysteine or tyrosine residues that may be essential for function.
In order to overcome these and other disadvantages, alternative activated PEGs have been introduced, such as succinimidyl succinate derivatives of mPEG (“SS-PEG”) (Abuchowski, A., et al. (1984) Cancer Biochem Biophys 7:175-186). Under mild conditions, SS-PEG reacts quickly with proteins (within 30 minutes), yielding active, yet extensively modified conjugates.
M. Saifer, et al., in U.S. Pat. No. 5,468,478, disclose polyalkylene glycol-mono-N-succinimidyl carbonates and conjugates produced therefrom. S. Zalipsky, in U.S. Pat. No. 5,612,460, discloses methods for preparation of poly(ethylene glycol)-N-succinimidyl carbonates. This form of the polymer (“SC-PEG”) reacts readily with the amino groups of proteins, as well as peptides of low molecular weight and other materials that contain free amino groups, with which it forms urethane bonds.
Urethane (or carbamate) linkages between the amino groups of the protein and the PEG are also known in the art to be produced from other PEG-carbonate derivatives (Beauchamp, C., et al. (1983) Anal Biochem 131:25-33; Veronese, F. M., et al. (1985) Appl Biochem Biotechnol 11:141-152). Reactive MPEG intermediates and methods for their use are also known in the art for the synthesis of PEG conjugates of bioactive components linked via amide bonds, ester bonds, secondary amines and thioester bonds, among others.
T. Suzuki et al. ((1984) Biochim Biophys Acta 788:248-255) covalently coupled immunoglobulin G (“IgG”) to mPEG that had been activated by cyanuric chloride. They studied the biological and physicochemical properties, such as antigen-binding activity and the molecular structure, size-exclusion chromatographic behavior, surface activity, interfacial aggregability and heat aggregability that induced nonspecific activation of complement by the PEG-IgG conjugates. The coupling of PEG to IgG increased the apparent Stokes radius and the surface activity of IgG and stabilized IgG to heating and/or the exposure to interfaces, while no structural denaturation of IgG was observed. The suppression of nonspecific aggregability was attributed mainly to the steric inhibition of the association between the PEGylated IgG molecules. These results indicated the utility of mPEG-coupled IgG as an intravenous preparation and also suggested the utility of PEG as an additive to stabilize unmodified IgG for intravenous use.
K. A. Sharp et al. ((1986) Anal Biochem 154:110-117) investigated the possibility of producing biospecific affinity ligands for separating cells in aqueous two-phase polymer systems on the basis of cell surface antigens. Rabbit anti-human erythrocyte IgG was reacted with cyanuric chloride-activated mPEGs with molecular weights of approximately 0.2, 1.9 and 5 kDa at various molar ratios of PEG to lysine groups on the protein. The partition coefficient of the protein in a two-phase system containing dextran and PEG increased with increasing degree of modification and increasing molecular weight of the MPEG. There was a concomitant loss in ability to agglutinate human erythrocytes.
R. H. Tullis, in U.S. Pat. No. 4,904,582, discloses oligonucleotide conjugates wherein the oligonucleotides are joined through a linking arm to a hydrophobic moiety, which could be a polyoxyalkylene group. The resulting conjugates are said to be more efficient in membrane transport, so as to be capable of crossing the membrane and effectively modulating a transcriptional system. In this way, the compositions can be used in vitro and in vivo for studying cellular processes, protecting mammalian hosts from pathogens, facilitating gene therapy, and the like.
Excessive conjugation of polymers and/or conjugation involving the active site of a therapeutic moiety where groups associated with bioactivity are found, however, can often result in loss of activity and, thus, in loss of therapeutic efficacy. This is often the case with lower molecular weight peptides that have few attachment sites that are not associated with bioactivity. For example, I. Benhar et al. ((1994) Bioconjug Chem 5:321-326) observed that PEGylation of a recombinant single-chain immunotoxin resulted in the loss of specific target immunoreactivity of the immunotoxin. The loss of activity of the immunotoxin was the result of attachment of PEG to two lysine residues within the antigen-combining region of the immunotoxin.
Although the covalent attachment of PAGs and PAOs (e.g., PEGs, PEOs, etc.) to therapeutic proteins is intended to eliminate their immunoreactivity, PEGylated proteins remain weakly immunogenic. This immunogenicity appears to be due, at least in part, to the fact that PEG and PAO polymers are themselves somewhat antigenic and immunogenic. For example, rabbits have been immunized to various PEGs by injecting the animals with conjugates in which PEG was coupled to an immunogenic carrier protein (Richter, A. W., et al. (1983) Int Arch Allergy Appl Immunol 70:124-131). In addition, a monoclonal antibody that reacts with the polyether backbone of PEG has been developed by injecting mice with an mPEG conjugate of β-glucuronidase and selecting a hybridoma clone that secretes an anti-PEG antibody (Cheng, T.-L., et al. (1999), Bioconjug. Chem. 10:520-528; Cheng, T.-L., et al. (2000), Bioconjug. Chem. 11:258-266; Tsai, N.-M., et al. (2001), Biotechniques 30:396-402; Roffler, S., et al., U.S. Pat. Nos. 6,596,849 and 6,617,118; the disclosures of all of which are incorporated herein by reference in their entireties). Another monoclonal antibody that reacts with the polyether backbone of PEG has been disclosed recently by Roberts, M. J., et al., in U.S. Patent Application No. 2003/001704 A1.
A number of investigators have disclosed the preparation of linear or branched “non-antigenic” PEG polymers and derivatives or conjugates thereof (see, e.g., U.S. Pat. Nos. 5,428,128; 5,621,039; 5,622,986; 5,643,575; 5,728,560; 5,730,990; 5,738,846; 5,811,076; 5,824,701; 5,840,900; 5,880,131; 5,900,402; 5,902,588; 5,919,455; 5,951,974; 5,965,119; 5,965,566; 5,969,040; 5,981,709; 6,011,042; 6,042,822; 6,113,906; 6,127,355; 6,132,713; 6,177,087, and 6,180,095; see also PCT publication WO 95/13090 and published U.S. patent application nos. 2002/0052443, 2002/0061307 and 2002/0098192). Most of the examples in the foregoing patents and patent applications employ polymers containing one or more strands of mPEG, e.g., di-mPEG-lysine. To date, however, there has been no disclosure of a mechanism for rendering the PEG in such polymers or conjugates non-antigenic.
Thus, there exists a need for the identification of methods of producing PAO-containing (e.g., PEG- and/or PEO-containing) conjugates, particularly conjugates between such water-soluble polymers and therapeutic proteins, with reduced, substantially reduced or no detectable antigenicity. Such conjugates will have the benefits provided by the polymer component of increased stability and bioavailability in vivo, but will not elicit a substantial immune response in an animal into which the conjugates have been introduced for therapeutic or diagnostic purposes.